Xu Hongjie, Wu Feng, Zhang Hongyu, Yang Chao, Li Kai, Wang Hailong, Yang Honghui, Liu Yue, Ding Bai, Tan Yingjun, Yuan Ming, Li Yinghui, Dai Zhongquan
State Key Laboratory of Space Medicine Fundamentals and Application, China Astronaut Research and Training Center, Beijing, China.
State Key Laboratory of Space Medicine Fundamentals and Application, China Astronaut Research and Training Center, Beijing, China.
Biochimie. 2017 Jul;138:184-193. doi: 10.1016/j.biochi.2017.04.015. Epub 2017 Apr 27.
Microgravity influences the activity of osteoblast, induces actin microfilament disruption and leads to bone loss during spaceflight. Mechanical stress such as gravity, regulates cell function, response and differentiation through dynamic cytoskeleton changes, but the mechanotransduction mechanism remains to be fully elucidated. Previous, we demonstrated actin microfilament mediated osteoblast Cbfa1 responsiveness to BMP2 under simulated microgravity (SMG). Here, we explored a potential molecular and its detailed mechanism of actin cytoskeleton functioning on BMP2-Smad signaling in MC3T3-E1 under SMG. Results showed that the actin microfilament-disrupting agent, cytochalasin B (CB), reduced BMP2-induced activation, translocation of Smad1/5/8 and Runx2 expression. SMG also inhibited BMP2-Smad signaling, which was rescued by actin cytoskeleton stabilizing agent, Jasplakinolide (JAS). Furthermore, we found that siRNA mediated knockdown of calponin 1 (CNN1), an actin binding protein, markedly promoted BMP2-Smad signaling and abolished both inhibition of CB, SMG on BMP2-Smad signaling and the rescue action of JAS. Overexpression of CNN1 inhibited the p-Smad induced by BMP2. Bidirectional Co-IP experiments demonstrated CNN1 could interacted with Smad or p-Smad protein. Furthermore, CB or SMG decreased the phosphorylated CNN1 and increased its interaction with Smad or p-Smad. Combined with the phosphorylation of CNN1 inhibites its actin binding activity, these results indicate that actin cytoskeleton depolymerization inhibites BMP2 signaling via blocking of Smad by dephosphorylated CNN1 in osteoblast cells. Thus, we provide new important insights into the mechanism of mechanotransduction under SMG condition, which probably contribute to bone formation decrease induced by SMG.
微重力会影响成骨细胞的活性，诱导肌动蛋白微丝破坏，并导致太空飞行期间的骨质流失。诸如重力这样的机械应力通过动态细胞骨架变化来调节细胞功能、反应和分化，但其机械转导机制仍有待充分阐明。此前，我们证明了在模拟微重力（SMG）条件下，肌动蛋白微丝介导成骨细胞Cbfa1对骨形态发生蛋白2（BMP2）的反应性。在此，我们探讨了在SMG条件下，肌动蛋白细胞骨架在MC3T3-E1细胞中对BMP2-Smad信号传导发挥作用的潜在分子及其详细机制。结果显示，肌动蛋白微丝破坏剂细胞松弛素B（CB）可降低BMP2诱导的激活、Smad1/5/8的易位以及Runx2的表达。SMG也会抑制BMP2-Smad信号传导，而肌动蛋白细胞骨架稳定剂茉莉酮酸甲酯（JAS）可挽救这一抑制作用。此外，我们发现小干扰RNA（siRNA）介导的肌动蛋白结合蛋白钙调蛋白1（CNN1）敲低显著促进了BMP2-Smad信号传导，并消除了CB和SMG对BMP2-Smad信号传导的抑制作用以及JAS的挽救作用。CNN1的过表达抑制了BMP2诱导的磷酸化Smad。双向免疫共沉淀（Co-IP）实验表明，CNN1可与Smad或磷酸化Smad蛋白相互作用。此外，CB或SMG会降低磷酸化的CNN1水平，并增加其与Smad或磷酸化Smad的相互作用。结合CNN1的磷酸化会抑制其肌动蛋白结合活性，这些结果表明，在成骨细胞中，肌动蛋白细胞骨架解聚通过去磷酸化的CNN1阻断Smad来抑制BMP2信号传导。因此，我们为SMG条件下的机械转导机制提供了新的重要见解，这可能有助于解释SMG诱导的骨形成减少现象。
